Expression and purification of native and truncated forms of CadF, an outer membrane protein of Campylobacter

International Journal of Biological Macromolecules
Laurent MamelliJean-Michel Bolla

Abstract

Campylobacter is now recognized as the most common bacterial agent of gastroenteritis. The adhesion of bacteria to intestinal cells is a major step in human colonization. The binding of Campylobacter jejuni cells to fibronectin (Fn), a component of the extra cellular matrix, is mediated by a 37,000 outer membrane protein termed CadF for Campylobacter adhesion to Fn. CadF protein is very hard to purify from Campylobacter membranes. In order to study the conformation of this protein, we set out to clone, express, purify, and re-fold the CadF protein. The nucleotide sequence encoding the N-terminal domain of the CadF protein was cloned in a pET-based expression vector. The recombinant protein was further produced in Escherichia coli, purified from inclusion bodies, and refolded. More specifically, the purification experiments were set-up as follows: (i) protein aggregates were collected from cell-lysates, solubilized in urea and enriched by ion-exchange chromatography; (ii) refolding was achieved by drop-by-drop dilution method in detergent containing buffer and monitored by CD measurements; (iii) the protein was finally purified to homogeneity by gel filtration chromatography. In spite of our success in purifying the N-terminal d...Continue Reading

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Citations

Jun 28, 2011·The Journal of Antimicrobial Chemotherapy·Manuella Catel-FerreiraEmmanuelle Dé
Sep 23, 2008·Applied and Environmental Microbiology·Kari D Shoaf-SweeneyMichael E Konkel
Nov 3, 2009·Journal of Bacteriology·Michael E KonkelRebecca C Flanagan
Apr 28, 2012·International Journal of Biological Macromolecules·Shi-Hui WangYan Sun
Jun 19, 2007·FEMS Microbiology Letters·Malgorzata Krause-GruszczynskaSteffen Backert
Dec 14, 2006·Proteomics·Laurent MamelliJean-Michel Bolla
Aug 19, 2007·Nature Reviews. Microbiology·Kathryn T YoungVictor J Dirita
Apr 25, 2020·Frontiers in Microbiology·Michael E KonkelCourtney M Klappenbach

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Campylobacteriosis (ASM)

Campylobacteriosis is caused by the bacteria Campylobacter jejuni and is a common cause of gastroenteritis in humans. Discover the latest research on Campylobacteriosis here.